Influenza virus replicated in mammalian cell culture and vaccine production

ABSTRACT

The invention provides replication of high growth influenza virus strains, derived from clinical isolates, in cultured mammalian cells by infecting the mammalian cells with the high growth strains to obtain infected cells, and culturing the cells while maintaining a trypsin concentration range of 0.05-1.0 μg/ml in the culture medium, where the resulting replicated virus is suitable for use in mammalian influenza vaccines and vaccination methods, which are also provided by the invention.

This application is a continuation of U.S. application Ser. No.08/628,384, filed Apr. 5, 1996, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 08/340,254, filed Nov.16, 1994, now abandoned which is a continuation-in-part of U.S.application Ser. No. 08/294,644, filed Aug. 23, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention, in the fields of virology and vaccine production,relates to replicated mammalian influenza viruses, grown in mammaliancell culture, which are suitable for use in mammalian influenza virusvaccine production. The replicated viruses are obtained from high growthstrains of (i) reassortants between high growth master donor strains andclinical isolates, or (ii) passaged clinical isolates. The infected cellculture uses a low concentration range of trypsin (0.05-1.0 μg/ml),continuously present in the medium, to provide high titers of thereplicated virus. The invention also relates to methods for making andusing such replicated viruses, such as for vaccine compositions and forvaccination methods.

2. Related Art

For the past several decades, fertilized chicken eggs have been used asa host system to replicate human influenza viruses with infectivitytiters sufficient for use in vaccine production. Clinical isolates ofhuman influenza virus are taken from infected patients and arereassorted in embryonated chicken eggs with laboratory-adapted masterstrains of high-growth donor viruses. The purpose of this reassortmentis to increase the yield of candidate vaccine strains achieved byrecombining at least the HA and NA genes from the primary clinicalisolate isolates, with the internal genes of the master strain donorviruses. The high growth reassortant vaccine strains must also notcontaminated with genes coding for antigenic determinants of thelaboratory adapted viruses. This provides high growth reassortantshaving antigenic determinants similar to those of the clinical isolates.(Robertson et al., Biologicals 20:213-220 (1992)). The reassortedinfluenza virus is then grown in embryonated chicken eggs, purified fromvirus-containing allantoic fluid of the eggs and subsequentlyinactivated for use as vaccines.

However, a large body of data now suggests that this is a problematicsystem because of the frequency of viral mutation in antigenic sites ofthe major virus glycoprotein, hemagglutinin (HA), during replication inthe chicken eggs. Even a single passage of a human influenza virusisolate or reassortant in chicken eggs leads to the selection of viralvariants that differ in their antigenic determinants from those of theoriginal clinical isolates. For example, the cultivation of influenza Aand B viruses in chicken eggs often leads to the selection by the hostsystem of variants having antigenic and structural changes in the viralHA molecule, making the variants ineffective or significantly lesseffective when used in an influenza vaccine (Kodihalli et al., J Virol.69:4888-4897 (1995); Gubareva et al., Virol. 199:89-97 (1994); Katz &Webster, J Infect. Dis. 160:191-198 (1989); Wood et al., Virol.171:214-221 (1989); Katz et al., Virology 156:386-395 (1987); Robertsonet al., Virology 143:166-174 (1985)). In addition, the replicativeproperties of egg-grown viruses are not as consistent with naturalinfection as those of viruses grown in mammalian cells (Katz et al., JVirol. 64:1808-1811 (1990); Robertson et al., Virology 179:35-40(1990)).

Additionally, embryonated chicken eggs have potentially seriouslimitations as a host system, e.g., due to the lack of reliableyear-around supplies of high-quality eggs and the low susceptibility ofsummer eggs to influenza virus infection (Monto, et al., J. Clin.Microb. 13:233-235 (1981)). Furthermore, the presence of adventitiousagents in eggs can jeopardize the preparation of live-attenuatedinfluenza virus vaccines. Adventitious agents are infectiouscontaminants (such as other viruses) in host systems that make themunsuitable or uncertifiable for use in vaccine production.

Cultured mammalian cells have also been used for virus replication andhave been classified into at least two distinct groups. Primary diploidcells are those derived from intact tissue and have not beensubcultivated. Continuous cell lines (CCLs) are cultured primary cellsthat replicate indefinitely and may be capable of growth in suspensionculture. Haylick, in Continuous Cell Lines as Substrates forBiologicals, Arlington, Va., p. 2 (1988).

At present, many viral vaccines other than influenza are produced usingprimary trypsinized cells, including cells from monkey kidneys, and thekidneys of rabbits and hamsters. Primary diploid cell cultures havecertain advantages such as easy preparation using simple media andbovine sera and sensitivity to a wide-range multiple viruses. However,primary diploid cells suffer from disadvantages, such as contaminationby various adventitous agents, variable quality and sensitivity; anddifficulty in obtaining suitable tissue for cultivation (e.g., monkeykidneys).

For example, primary diploid cell cultures obtained from monkey kidneysof wild animals usually contain endogenous viruses (Grachev, InBurgasov; ed., "Guidance for the Production of Vaccines and Sera."Medicine, Moscow, p 176 (1978)). The number of adventitous agentsdepends on many factors, such as the methods of isolation, the cellsystems used, the number of passages, the time of incubation andco-cultivation. The frequency of isolation of viruses from primarydiploid cell cultures of monkey kidneys is directly proportional to theincubation period of the cells. Grachev, In Zh. Microbiol. Epidemiol.Immunobiol. 2:76 (1987).

In contrast, the advantages of using continuous cell lines are theirretention of original antigenic characteristics of the infected virus;standardization, high susceptibility to variants of the same virus, andability to be grown as a large mass of cells using microcarrier orsuspension fermentor systems.

However, these advantages themselves do make such cell lines suitablefor use in vaccine production. Mizrahi, ed., Viral Vaccines, Wiley-Liss,New York (1990), pp. 39-67. For example, influenza A viruses isolatedand passaged exclusively in mammalian cell cultures have been found insome cases to retain most or all of their original antigeniccharacteristics, a feature that would prove highly advantageous invaccine production. (Katz et al., Virology 165:446-456 (1988); Robertsonet al., Virology 179:35-40 (1990); Katz et al., J. Infect. Dis.160:191-198 (1989); Wood et al., Virology 171:214-221 (1989)).

However, mammalian primary diploid cell cultures present difficulties asa host system for vaccine production. This is due to problems such ascontamination of the cell culture with adventitious agents, variablequality of the cells in the cell culture, different sensitivities of thecells to variants of the same virus, low virus titers and the high costand difficulties in obtaining and preparing such cell cultures. Inanother example, although human diploid (MRC-5) cells can support thegrowth of influenza viruses, such systems have stringent growth mediarequirements making them suboptimal for large-scale production ofinfluenza viruses for use in vaccines.

Furthermore, only MDCK cells, among the continuous cell lines tested,have been reported to support potentially sufficient growth andisolation of viruses (Frank et al., J. Clin. Microb. 10:32-36 (1979);Schepetink & Kok, J. Virol. Methods 42:241-250 (1993)). However, thisline has been found to produce tumors and has thus not been certifiedfor vaccine production, as not substantially free of adventitiousagents.

Two other continuous cell lines--African green monkey kidney (Vero)cells and baby hamster kidney (BK-21)--are characterized, approved andcertified by the World Health Organization (WHO) for production of humanvaccines. However, Vero cells, while certified, were previously foundunsuitable for large-scale production of human influenza virus vaccines.For example, the growth of influenza B in Vero cells was greatlyrestricted as compared to MDCK cells (Nakamura et al., J. Gen. Virol.56:199-202 (1981)). Additionally, attempts to use Vero cells to evaluatethe rimantadine sensitivity of human H1N1 and H3N2 influenza A virusesgave ambiguous results, due to the low titers of viruses produced inthese cells, as compared with MDCK cells (Valette et al., Antimicrobiol.Agent and Chemotherapy 37:2239-2240 (1993)).

Thus, these and other studies indicate that influenza viruses have notpreviously replicated well in Vero cells, making them unsuitable forlarge-scale vaccine production. (Demidova et al., Vopr. Virosol(Russian) 346-352 (1979); Lau & Scholtissek, Virology 212:225-231(1995)).

Itoh et al., Japan J. Mol. Sci. Biol. 23:227-235 (1970) discloses astudy of Vero cell cultures for infection with parainfluenza andinfluenza A viruses using a final added trypsin concentration of 1.5-1.8μg/ml. Itoh used chicken egg-grown viruses (Itoh et al. Virus 18:214-226(1968)) to infect Vero cells under the above conditions, and foundenhanced viral titers (over non-trypsin containing medium) for some ofthe influenza A strains tested, but Itoh could not demonstrate enhancedviral titers for influenza B strains.

Influenza viruses have eight negative-sense RNA (nsRNA) gene segmentsthat encode at least 10 polypeptides, including RNA-directed RNApolymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase(NA), hemagglutinin (HA, active with cleavage and association ofsubunits HA1 and HA2), the matrix proteins (M1 and M2) and thenon-structural proteins (NS1 and NS2) (Krug et al., "Expression andReplication of the Influenza Virus Genome," in The Influenza Viruses, R.M. Krug, ed., Plenum Press, New York (1989), pp. 89-152).

Influenza viruses are enclosed by lipid envelopes, derived from theplasma membrane of the host cell. The HA and NA proteins are the primaryantigenic determinants and are embedded in the viral envelope bysequences of hydrophobic amino acids (Air et al., Structure, Function,and Genetics 6:341-356 (1989); Wharton et al., "Structure, Function, andAntigenicity of the Hemagglutinin of Influenza Virus" in The InfluenzaViruses, R. M. Krug, ed., Plenum Press, New York (1989), pp. 153-174).

The major influenza virus glycoprotein, HA, is synthesized in infectedcells as a single polypeptide. Post-translational protease cleavage ofthe precursor HA results in the formation of the two subunits, HA1 andHA2, joined by a disulfide bond. Cleavage is essential for production ofinfectious viruses: virions containing uncleaved HA are noninfectious.The cleavage process can occur intracellularly or extracellularly. WhileHAs of infectious viruses are cleaved by extracellular proteases, suchas from intestinal bacteria or the pancreas in vivo, the HAs of human,swine and most avian influenza virus strains cannot be cleaved byintracellular proteases. Therefore, replication of these viruses in manycell cultures requires the addition of a protease (such as trypsin) tothe maintenance medium to ensure HA cleavage, thereby permittingactivation of the progeny virus so that the rounds of infection cancontinue. Previous publications have suggested using a trypsinconcentration in the range of 4-25 μg/ml (U.S. Pat. No. 4,500,513). Verocells were recently discovered to produce an activity that inactivatesexogenous trypsin (Kaverin & Webster, J. Virol. 69:2700-2703 (1995)).

The background art has thus established a long-felt need for a method ofinfluenza virus and vaccine production in a host cell system thatimproves on the use of chicken eggs. Such a host system would maintainantigenic properties of the clinical isolates of the natural virus andprovide high titers of replicated virus, without having significantadventitious agents which are unsuitable for vaccine production.

SUMMARY OF THE INVENTION

The present invention includes methods, replicated viruses and vaccinecompositions using high growth strains of mammalian influenza viruses,passaged from, or reassorted with, viral clinical isolates. The highgrowth strains of the invention can be reassortants made with laboratoryhigh growth master donor strains, or they can be isolates that have beenpassaged in primary cell culture and selected for high growth. Thesemethods use mammalian host cells that are infected with the high growthstrains and then cultured with a continuous and low trypsinconcentration in the culture medium. The methods provide replicatedvirus having high infectivity titers. These replicated, high growthstrains are suitable for, and included in, influenza virus vaccines ofthe invention, for which the replicated virus is inactivated and/orattenuated.

The invention thus provides replicated influenza viruses and vaccinesthat comprise at least one replicated influenza virus strain, where thevaccine contains the replicated virus in an inactivated or attenuatedform. These vaccines have substantially similar antigenicity to theviral clinical isolates, relative to chicken egg-grown viruses, whereselection pressures in the eggs change the viruses' antigenicity fromthat of the clinical isolates, such as through mutation of the HA gene.

In contrast to influenza viruses grown in other host cell types, such aschicken eggs, replicated influenza viruses and vaccines of the presentinvention provide at least one of: (i) substantially similarantigenicity to the clinical isolate; (ii) consistently high titers;(iii) lack of contamination by adventitious agents; (iv) consistent cellgrowth qualities; and (v) relatively less cost and technicaldifficulties in host cell replication.

Influenza virus vaccines of the invention can include at least onereplicated virus strain (e.g., 1-50 strains) of a mammalian influenzavirus A or B. Preferably the mammal host cells are continuous cellcultures of primary, cultured epithelial cells or fibroblasts, asmammalian cell lines of passage number 10-250. Preferably used forvaccine production are primary Vero cells as a continuous cell cultureof a passage number of about 20-250. Currently available and certified(e.g., by the World Health Organization, WHO). Vero cell lines arepassage number 135-190 (e.g., ATTC NO:X38).

Replicated influenza virus of the invention, in isolated, purified orconcentrated form, preferably has an infectivity titer of about 10⁶ -10⁹(such as 10⁶ -10⁷, 10⁷ and 10⁸ -10⁹, or any range or value therein)plaque forming units (PFU) per ml.

It is now discovered that providing a continuous, low trypsinconcentration in the cell culture can circumvent the problem of trypsininactivation or viral replication inhibition found previously inmammalian cell cultures. Such trypsin concentrations are also discoveredto ensure multicycle replication that is comparable, in some or allrespects, to that seen with either human influenza A or B viruses grownin chicken eggs.

The present invention also provides vaccine compositions comprising atleast one strain of a replicated influenza virus of the presentinvention, in inactivated or attenuated form, optionally furthercomprising at least one of: (a) at least one pharmaceutically acceptablecarrier or diluent; (b) at least one adjuvant and/or (c) at least oneviral chemotherapeutic agent. The at least one carrier, diluent,adjuvant or chemotherapeutic agent enhances at least one immune responseto at least one pathogenic influenza virus in a mammal administered thevaccine composition.

The present invention also provides a method for eliciting an immuneresponse to at least one influenza virus strain in a mammal, whichresponse is prophylactic or therapeutic for an influenza virusinfection. The method comprises administering to the mammal a vaccinecomposition comprising an inactivated and/or attenuated, replicatedinfluenza virus of the present invention. The composition is provided inan amount that is protective or therapeutic for the mammal against aclinical influenza virus pathology caused by infection with at least oneinfluenza A or B virus strain.

Other objects, features, advantages, utilities and embodiments of thepresent invention will be apparent to skilled practitioners from thefollowing detailed description and examples relating to the presentinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D: Analysis of the abundance of sialic acid (SA) α2,3-Galactose(Gal), and SAα2,6-Gal linkages on the surface of Vero and MDCK cells.The profile shown depicts cell number as a function of the log ofrelative fluorescence intensity of SAα2,3-Gal-α(2,3; Maackia amurensisagglutinin) and SAα2, 6-Gal α(2, 6; Sambucus nigra agglutinin)--specificlectin-reactive oligosaccharide expression on the surface of: Vero cells(FIGS. 1A-1B) and MDCK cells (FIGS. 1C-1D).

FIGS. 2A-B. Protein synthesis in Vero and MDCK cells infected with (FIG.2A) influenza A/England/1/53 (H1N1) or (FIG. 2B) influenza B/AnnArbor/1/86 viruses. Vero or MDCK cells were infected with eitherinfluenza A or B viruses at a multiplicity of infection (m.o.i.) of ˜30PFU/cell. After absorption for 1 hour at 37° C., the cells were washedand incubated for 5 hours (for influenza A virus) or 7 hours (forinfluenza B virus). Thereafter the cells were radioactively labeled withTran(³⁵ S)methionine/cysteine (100 μCi/ml) for 2 hours at 37° C. Cellswere washed, lysed and immunoprecipitated with specific monoclonalantibodies against HA, NP, M1, M2 and NS1 proteins. Uninfected cells,cells infected with either influenza A strain (FIG. 2A) or B strain(FIG. 2B) and cell precipitates were analyzed by SDS-PAGE. The positionsof viral proteins HA, NP, M1, NS1, M2 and NS2 are indicated.

FIGS. 3A-F. Electron micrographs showing influenza A/England/1/53 (H1N1)virus infected Vero and MDCK cells. The virions that budded from theapical surface are shown for MDCK cells (FIG. 3A) and Vero cells (FIG.3B). The nuclear breakdown (FIG. 3C, FIG. 3D) and cytoplasmic blebbing(FIG. 3E, FIG. 3F) typical of apoptotic cells is also observed with MDCKcells (FIG. 3C, FIG. 3E) and Vero cells (FIG. 3D, FIG. 3F). "v",virions; "b", blebbing of nuclear envelope. Magnification is ×9,250.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides replicated influenza virus usingcontinuous cell lines of cultured mammalian primary cells, in mediahaving a low continuous trypsin concentration. The viruses used forgenerating the replicated virus are high growth strains of clinicalisolates of at least one mammalian influenza virus strain. The highgrowth strains are selected from passaged or reassorted clinicalisolates. The use of low trypsin concentrations (e.g., 0.05-1.0 μg/ml)is unexpectedly discovered to provide high infectivity titers ofreplicated influenza virus having substantially similar antigenicity tothat of the clinical isolates, where the host cells and replicatedviruses are suitable for use in influenza virus vaccine production. Suchreplicated viruses have improved antigenic stability as compared withviruses grown in embryonated chicken eggs, while also being certifiablefor human vaccine production.

The term "high growth" as used in the literature can be ambiguous as itcan refer to either high HA titers or high infectivity titers, assayedin either eggs or tissue culture. In the context of the presentinvention, "high growth" viruses are defined to produce high infectivitytiters in in vitro tissue culture replication systems, such as 10⁵ -10¹⁰PFU/ml, and preferably 10⁶ -10⁹ PFU/ml.

The screening of influenza viruses for use in replication or vaccineproduction, can be assayed using any known and/or suitable assay, as isknown in the art. Such assays (alone or in any combination) that aresuitable for screening include, but are not limited to, viralreplication, quantitative and/or qualitative measurement of inactivation(e.g., by antisera), transcription, replication, translation, virionincorporation, virulence, HA or NA activity (HA activity preferred),viral yield, and/or morphogenesis, using such methods as reversegenetics, reassortment, complementation, and/or infection. For example,virus replication assays can be used to screen for attenuation orinactivation of the virus. See, e.g., Krug, R. M., ed., The InfluenzaViruses, Plenum Press, New York, (1989).

For culturing mammalian host cells used for viral replication in methodsof the present invention, a trypsin concentration between 0.05 and 1.0μg/ml (e.g., 0.05-0.09, 0.1-0.2, 0.2-0.5, 0.6-0.9, 0.7-0.9, 0.8-1.0 orany range or value therein) can be used during the host cell growthcycle and/or viral growth cycle, by adding trypsin continuously or atintervals to the culture medium. The multiplicity of infection of thevirus for the host cell culture can be from 1×10⁻⁶ TCID₅₀ to 5×10⁻³TCID₅₀ per cell (e.g., 1×10⁻⁶ TCID₅₀ to 1×10⁻⁵ TCID₅₀, 2×10⁻⁵ TCID₅₀ to1×10⁻⁴ TCID₅₀, or 2×10⁻⁴ TCID₅₀ to 5×10⁻³ TCID₅₀, or any range or valuetherein. "TCID₅₀ " stands for the tissue culture infective dose, or thedose sufficient to infect 50% of the cells.

Cell Lines and Influenza Viruses That Can Be Used in the PresentInvention

According to the present invention, any clinical isolate of at least onestrain of a mammalian influenza A or B virus can be used to obtain highgrowth strains suitable for replicating in mammal host cells, in orderto provide replicated influenza virus of the invention. The clinicalisolate can be made into a high growth strain by reassortment with ahigh growth master donor strain, or by multiple passages of the clinicalisolate in continuous mammalian cell lines, with selection of highgrowth variants.

The clinical isolates are preferably reassorted with laboratory highgrowth master donor strains in culture, and the reassortants selectedthat have HA and NA genes from the isolates, and internal genes from thehigh growth master laboratory strains. For example, the resulting strainfor the influenza A component can be a reassortant virus that containsinternal genes from the master donor strain A/PR/8/34 (H1N1), whichprovides high growth in host cells, as well as at least the HA genecoding for at least one surface antigen of the clinical isolate of theinfluenza virus (using known methods, e.g., according to Robertson etal., Biologicals 20:213-220 (1992)). Such reassortants can be made morerapidly than high growth strains made by multiple passages of theclinical isolates.

A high growth virus strain, derived from the clinical isolate strain ora reassortant thereof, is then replicated in suitable mammalian hostcells in the continuous presence of a trypsin concentration of 0.05-1.0μg/ml, to obtain sufficiently high infectivity titers (e.g., 10⁶ -10⁹PFU/ml) that are useful for vaccine production.

Cell Lines

According to methods for replicating viruses of the present invention,suitable mammalian host cells can be used, including Vero cells or othermammalian cells suitably excluding adventitious agents, preferably of asuitable passage number that can be certified according to the WHOrequirements for vaccine production (Mizrahi, ed., Viral Vaccines,Wiley-Liss, New York (1990), pp. 39-60). Non-limiting examples of celllines that can be suitable for methods, viruses and compositions used inthe present invention, include, but are not limited to, mammalianfibroblast or cultured epithelial cells as continuous cell lines.Further non-limiting examples include Vero, MDBK, BK-21 and CV-1 cells,readily available from commercial sources (e.g., ATCC, Rockville, Md.).Vero cells of passage number less than 191 are preferred, or any rangeor value therein.

Continuous Cell Lines

Continuous cell lines (CCLs), derived from primary diploid cells, arepreferred for replicating influenza virus according to the presentinvention. CCLs possess advantages over primary diploid cells, such assuitability for the large-scale cultivation; high sensitivity todifferent viral variants; unrestricted and stable growth; and low cost(relative to primary diploid cell cultures). Montagnon et al., Dev.Biol. Stand. 47:55 (1987); Grachev, Virol. 4:44 (1983); Smith et al., J.Clin. Microbiol. 24:265 (1986); Grachev et al., in Guidance for theProduction of Vaccines and Sera, Burgamov, ed., Medicine, Moscow p. 176(1978)).

WHO certified, or certifiable, continuous cell lines are preferred forproducing influenza virus vaccines of the present invention. Therequirements for certifying such cell lines include characterizationwith respect to at least one of genealogy, growth characteristics,immunological markers, virus susceptibility tumorigenicity and storageconditions, as well as by testing in animals, eggs, and cell culture.Such characterization is used to confirm that the CLLs are free fromdetectable adventitious agents. In some countries, karyology may also berequired. In addition, tumorigenicity is preferably tested in cells thatare at the same passage level as those used for vaccine production. Thereplicated virus is preferably purified by a process that has been shownto give consistent results, before being inactivated or attenuated forvaccine production (see, e.g., World Health Organization TRS No. 673(1982)).

It is preferred to establish a complete characterization of thecontinuous cell line to be used, so that appropriate tests for purity ofthe final product can be included. Data that can be used for thecharacterization of a continuous cell line to be used in the presentinvention includes (a) information on its origin, derivation, andpassage history; (b) information on its growth and morphologicalcharacteristics; (c) results of tests of adventitious agents; (d)distinguishing features, such as biochemical, imunological, andcytogenetic patterns which allow the cells to be clearly recognizedamong other cell lines; and (e) results of tests for tumorigenicity.Preferably, the passage level, or population doubling, of the cell lineused is as low as possible.

It is preferred that the replicated virus produced in continuous celllines is highly purified prior to vaccine formulation according to theinvention. Generally, the purification procedures will result in theextensive removal of cellular DNA, other cellular components, andadventitious agents. Procedures that extensively degrade or denature DNAcan also be used. See, e.g., Mizrahi, ed., Viral Vaccines, Wiley-Liss,New York pp. 39-67 (1990).

Vaccines

The resulting replicated virus can then be concentrated and/or purified(e.g., by centrifugation or column chromatography) and then inactivatedor attenuated using known method steps.

Inactivated Vaccines. Inactivated influenza virus vaccines of theinvention are provided by inactivating replicated virus of the inventionusing known methods, such as, but not limited to, formalin orβ-propiolactone treatment. Inactivated vaccine types that can be used inthe invention can include whole-virus (WV) vaccine or subvirion (SV)virus vaccine. The WV vaccine contains intact, inactivated virus, whilethe SV vaccine contains purified virus disrupted with detergents thatsolubilize the lipid-containing viral envelope, followed by chemicalinactivation of residual virus.

In addition, vaccines that can be used include those containing theisolated HA and NA surface proteins, which are referred to as surfaceantigen vaccines. In general, the responses to SV and surface antigen(i.e., purified HA or NA) vaccines are similar. An experimentalinactivated WV vaccine containing an NA antigen immunologically relatedto the epidemic virus and an unrelated HA appears to be less effectivethan conventional vaccines (Ogra et al., J. Infect. Dis. 135:499-506(1977)). Inactivated vaccines containing both relevant surface antigensare preferred.

Live Attenuated Virus Vaccines. Live, attenuated influenza virusvaccines, using replicated virus of the invention, can also be used forpreventing or treating influenza virus infection, according to knownmethod steps: Attenuation is preferably achieved in a single step bytransfer of attenuating genes from an attenuated donor virus to areplicated isolate or reassorted virus according to known methods (see,e.g., Murphy, Infect. Dis. Clin. Pract. 2:174-181 (1993)). Sinceresistance to influenza A virus is mediated by the development of animmune response to the HA and NA glycoproteins, the genes coding forthese surface antigens must come from the reassorted viruses or highgrowth clinical isolates. The attenuating genes are derived from theattenuated parent. In this approach, genes that confer attenuationpreferably do not code for the HA and NA glycoproteins. Otherwise, thesegenes could not be transferred to reassortants bearing the surfaceantigens of the clinical virus isolate.

Many donor viruses have been evaluated for their ability to reproduciblyattenuate influenza viruses. As a non-limiting example, the A/AnnArbor(AA)/6/60 (H2N2) cold adapted (ca) donor virus can be used forattenuated vaccine production (see, e.g., Edwards, J. Infect. Dis.169:68-76 (1994); Murphy, Infect. Dis. Clin. Pract. 2:174-181 (1993)).Additionally, live, attenuated reassortant virus vaccines can begenerated by mating the ca donor virus with a virulent replicated virusof the invention. Reassortant progeny are then selected at 25° C.(restrictive for replication of virulent virus), in the presence of anH2N2 antiserum, which inhibits replication of the viruses bearing thesurface antigens of the attenuated A/AA/6/60 (H2N2) ca donor virus.

A large series of H1N1 and H3N2 reassortants have been evaluated inhumans and found to be satisfactorily: (a) infectious, (b) attenuatedfor seronegative children and immunologically primed adults, (c)immunogenic and (d) genetically stable. The immunogenicity of the careassortants parallels their level of replication. Thus, the acquisitionof the six transferable genes of the ca donor virus by new wild-typeviruses has reproducibly attenuated these viruses for use in vaccinatingsusceptible adults and children.

Other attenuating mutations can be introduced into influenza virus genesby site-directed mutagenesis to rescue infectious viruses bearing thesemutant genes. Attenuating mutations can be introduced into non-codingregions of the genome, as-well as into coding regions. Such attenuatingmutations can also be introduced into genes other than the HA or NA,e.g., the PB2 polymerase gene (Subbarao et al., J. Virol. 67:7223-7228(1993)). Thus, new donor viruses can also be generated bearingattenuating mutations introduced by site-directed mutagenesis, and suchnew donor viruses can be used in the production of live attenuatedreassortants H1N1 and H3N2 vaccine candidates in a manner analogous tothat described above for the A/AA/6/60 ca donor virus. Similarly, otherknown and suitable attenuated donor strains can be reassorted withreplicated influenza virus of the invention to obtain attenuatedvaccines suitable for use in the vaccination of mammals. (Ewami et al.,Proc. Natl. Acad. Sci. USA 87:3802-3805 (1990); Muster et al., Proc.Natl. Acad. Sci. USA 88:5177-5181 (1991); Subbarao et al., J. Virol.67:7223-7228 (1993); U.S. patent application Ser. No. 08/471,100, whichreferences are entirely incorporated by reference)

It is preferred that such attenuated viruses maintain the genes from thereplicated virus that encode antigenic determinants substantiallysimilar to those of the original clinical isolates. This is because thepurpose of the attenuated vaccine is to provide substantially the sameantigenicity as the original clinical isolate of the virus, while at thesame time lacking infectivity to the degree that the vaccine causesminimal chance of inducing a serious pathogenic condition in thevaccinated mammal.

The replicated virus can thus be attenuated or inactivated, formulatedand administered, according to known methods, as a vaccine to induce animmune response in a mammal. Methods are well-known in the art fordetermining whether such attenuated or inactivated vaccines havemaintained similar antigenicity to that of the clinical isolate or highgrowth strain derived therefrom. Such known methods include the use ofantisera or antibodies to eliminate viruses expressing antigenicdeterminants of the donor virus; chemical selection (e.g., amantadine orrimantidine); HA and NA activity and inhibition; and DNA screening (suchas probe hybridization or PCR) to confirm that donor genes encoding theantigenic determinants (e.g., HA or NA genes) are not present in theattenuated viruses. See, e.g., Robertson et al., Giornale di Igiene eMedicina Preventiva 29.4-58 (1988); Kilbourne, Bull. M2 World HealthOrg. 41:643-645 (1969); Aymard-Henry et al., Bull. World Health Org.481:199-202 (1973); Mahy et al., J. Biol. Stand. 5:237-247 (1977);Barrett et al., Virology: A Practical Approach, Oxford IRL Press,Oxford, pp. 119-150 (1985); Robertson et al., Biologicals 20:213-220(1992).

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention, suitable forinoculation or for parenteral or oral administration, compriseattenuated or inactivated mammalian influenza viruses, optionallyfurther comprising sterile aqueous or non-aqueous solutions,suspensions, and emulsions. The composition can further compriseauxiliary agents or excipients, as known in the art. See, e.g., Berkowet al., eds., The Merck Manual, 15th edition, Merck and Co., Rahway,N.J. (1987); Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y. (1990); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987); Osol, A., ed.,Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa. pp.1324-1341 (1980); Katzung, ed. Basic and Clinical Pharmacology, FifthEdition, Appleton and Lange, Norwalk, Conn. (1992), which references andreferences cited therein, are entirely incorporated herein by referenceas they show the state of the art.

A virus vaccine composition of the present invention can comprise fromabout 10² -10⁹ plaque forming units (PFU)/ml, or any range or valuetherein, where the virus is attenuated. A vaccine composition comprisingan inactivated virus can comprise an amount of virus corresponding toabout 0.1 to 200 μg of hemagglutinin protein/ml, or any range or valuetherein.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and/or emulsions, which may containauxiliary agents or excipients known in the art. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Carriers or occlusive dressings can be used to increase skinpermeability and enhance antigen absorption. Liquid dosage forms fororal administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents. See, e.g., Berkow, infra,Goodman, infra, Avery's, infra, Osol, infra and Katzung, infra, whichare entirely incorporated herein by reference, included all referencescited therein.

When a vaccine composition of the present invention is used foradministration to an individual, it can further comprise salts, buffers,adjuvants, or other substances which are desirable for improving theefficacy of the composition. Adjuvants are substances that can be usedto augment a specific immune response. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the mammal beingimmunized. Examples of materials suitable for use in vaccinecompositions are provided in Osol, A., ed., Remington's PharmaceuticalSciences, Mack Publishing Co, Easton, Pa. (1980), pp. 1324-1341, whichreference is entirely incorporated herein by reference.

Heterogeneity in the vaccine may be provided by mixing replicatedinfluenza viruses for at least two mammalian influenza virus strains,such as 2-50 strains or any range or value therein. Influenza A or Bvirus strains having a modem antigenic composition are preferred.According to the present invention, vaccines can be provided forvariations in a single strain of an influenza virus or for more than onestrain of influenza viruses, using techniques known in the art.

A pharmaceutical composition according to the present invention mayfurther or additionally comprise at least one viral chemotherapeuticcompound, including, but not limited to, gamma globulin, amantadine,guanidine, hydroxybenzimidazole, interferon-α, interferon-β,interferon-γ, thiosemicarbarzones, methisazone, rifampin, ribavirin, apyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid,acyclovir, dideoxynucleosides, a protease inhibitor, or ganciclovir.See, e.g., Katzung, infra, and the references cited therein on pages798-800 and 680-681, respectively, which references are herein entirelyincorporated by reference.

The vaccine can also contain variable but small quantities of endotoxin,free formaldehyde, and preservative, which have been found safe and notcontributing to the reactogenicity of the vaccines for humans.

Pharmaceutical Purposes

The administration of the vaccine composition (or the antisera that itelicits) may be for either a "prophylactic" or "therapeutic" purpose.When provided prophylactically, the compositions are provided before anysymptom of influenza viral infection becomes manifest. The prophylacticadministration of the composition serves to prevent or attenuate anysubsequent infection. When provided therapeutically, the attenuated orinactivated viral vaccine is provided upon the detection of a symptom ofactual infection. The therapeutic administration of the compound(s)serves to attenuate any actual infection. See, e.g., Berkow, infra,Goodman, infra, Avery, infra and Katzung, infra, which are entirelyincorporated herein by reference, including all references citedtherein.

An attenuated or inactivated vaccine composition of the presentinvention may thus be provided either before the onset of infection (soas to prevent or attenuate an anticipated infection) or after theinitiation of an actual infection.

A composition is said to be "pharmacologically acceptable" if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a "therapeutically effective amount" if theamount administered is physiologically significant. A vaccine orcomposition of the present invention is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient that enhances at least one primary or secondaryhumoral or cellular immune response against at least one strain of aninfectious influenza virus.

The "protection" provided need not be absolute, i.e., the influenzainfection need not be totally prevented or eradicated, if there is astatistically significant improvement compared with a control populationor set of patients. Protection may be limited to mitigating the severityor rapidity of onset of symptoms of the inlfuenza virus infection.

Pharmaceutical Administration

A vaccine of the present invention may confer resistance to one or moreinfluenza strains by either passive immunization or active immunization.In active immunization, an inactivated or attenuated live vaccinecomposition is administered prophylactically, according to a method ofthe present invention. In another embodiment as passive immunization,the vaccine is provided to a host (i.e. a mammal), and the elicitedantisera is recovered and administered to a recipient suspected ofhaving an infection caused by at least one influenza virus strain.

In a second embodiment, the vaccine is provided to a mammalian female(at or prior to pregnancy or parturition), under conditions of time andamount sufficient to cause the production of antisera which serve toprotect both the female and the fetus or newborn (via passiveincorporation of the antibodies across the placenta or in the mother'smilk).

The present invention thus includes methods for preventing orattenuating infection by at least one influenza virus strain. As usedherein, a vaccine is said to prevent or attenuate a disease if itsadministration results either in the total or partial attenuation (i.e.,suppression) of a symptom or condition of the disease, or in the totalor partial immunity of the individual to the disease.

At least one inactivated or attenuated influenza virus, or compositionthereof, of the present invention may be administered by any means thatachieve the intended purpose, using a pharmaceutical composition aspreviously described.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, oral or transdermal routes.Parenteral administration can be by bolus injection or by gradualperfusion over time. A preferred mode of using a pharmaceuticalcomposition of the present invention is by intramuscular or subcutaneousapplication. See, e.g., Berkow, infra, Goodman, infra, Avery, infra andKatzung, infra, which are entirely incorporated herein by reference,including all references cited therein.

A typical regimen for preventing, suppressing, or treating an influenzavirus related pathology, comprises administration of an effective amountof a vaccine composition as described herein, administered as a singletreatment, or repeated as enhancing or booster dosages, over a period upto and including between one week and about 24 months, or any range orvalue therein.

According to the present invention, an "effective amount" of a vaccinecomposition is one that is sufficient to achieve a desired biologicaleffect. It is understood that the effective dosage will be dependentupon the age, sex, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect wanted. The ranges of effective doses provided below are notintended to limit the invention and represent preferred dose ranges.However, the most preferred dosage will be tailored to the individualsubject, as is understood and determinable by one of skill in the art,without undue experimentation. See, e.g., Berkow et al., eds., The MerckManual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman et al.,eds., Goodman and-Gilman's The Pharmacological Basis of Therapeutics,8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co.,Boston, Mass. (1985); and Katsung, infra, which references andreferences cited therein, are entirely incorporated herein by reference.

The dosage of an attenuated virus vaccine for a mammalian (e.g., human)adult can be from about 10³ -10⁷ plaque forming units (PFU)/kg, or anyrange or value therein. The dose of inactivated vaccine can range fromabout 1 to 50 μg of hemagglutinin protein. However, the dosage should bea safe and effective amount as determined by conventional methods, usingexisting vaccines as a starting point.

The dosage of immunoreactive HA in each dose of replicated virus vaccinecan be standardized to contain a suitable amount, e.g., 1-50 μg or anyrange or value therein, or the amount recommended by the U.S. PublicHealth Service (PHS), which is usually 15 μg, per component for olderchildren ≧3 years of age, and 7.5 μg per component for children <3 yearsof age. The quantity of NA can also be standardized, however thisglycoprotein can be labile during the process of purification andstorage (Kendal et al., Infect. Immun. 29:966-971 (1980); Kerr et al.,Lancet 1:291-295 (1975)). Each 0.5-ml dose of vaccine preferablycontains approximately 1-50 billion virus particles, and preferably 10billion particles.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples providedby way of illustration, and are not intended to be limiting of thepresent invention.

EXAMPLE 1 Reduced Production of Influenza Virus in Vero Cell Cultures isdue to Loss of Trypsin in the Culture Medium

Material & Methods

Cells. The Vero cell line (ATCC No. X38) (WHO-approved) was obtainedfrom the American Type Culture Collection (ATCC) at the 134th passage.The cells were cultivated as monolayers in 250 cm³ flasks at 37° C. and5% CO₂ in a growth medium of Eagles minimal essential medium (MEM)supplemented with 10% unheated fetal calf serum. For the growth ofMadin-Darby canine kidney (MDCK) cells and rhesus monkey kidney(LLC-MK2) cells, the medium used was MEM with 5% fetal calf serum heated30 minutes at 56° C. For the cultivation of a swine kidney cell line(SwK), RPMI 1640 medium was used with 5% heated fetal calf serum. Forthe experiments involving infection or mock-infection, the cells weregrown either in 50 cm³ flasks or in 6 well, 24-well and 96-well plates(Falcon Labware). Cell monolayers were washed three times with PBS andoverlaid with maintenance medium. The latter had the same composition asthe growth medium for each cell line, the serum being omitted and 0.3%bovine serum albumin (BSA) added. Unless otherwise stated, themaintenance medium contained L-1-tosylamide-2-phenylethyl chloromethylketone (TPCK) trypsin (Worthington Diagnostics, Freehold, N.J.) at 1.0μg/ml. Plaque assays were performed with TPCK trypsin (2.5 μg/ml).

Viruses. Vero-adapted influenza A/England/1/53 (H1N1) High Growth (HG),A/FW/1/50 (H1N1), and reassortant X-31 (H3N2) obtained from the parentsA/Aichi/2/68 X (B3N2) PR/8/34 (H1N1) viruses were used. The viruses werepassaged 5 times in Vero cell cultures, and the final stock viruspreparations contained 10⁷.3 to 10⁸.25 TCID₅₀ /0.2 ml and 32 to 128 HAU.In the preliminary experiments, the Vero-adapted A/Rome/49 (H1N1) strainwas used (10⁶.7 TCID₅₀ /0.2 ml, 16 to 32 HAU). HA and infectivitytitration were performed essentially as described in "AdvancedLaboratory Techniques for Influenza Diagnosis" (Immunol. Ser. 6, pp.51-57 (1975)). HA titrations were done in microtiter plates. Infectivitywas measured by an end point titration technique in MDCK cells grown in96-well plates with cytopathic effect (CPE) evaluation at 72 hourspostinfection (Table 1).

Results: Restoration of Multicycle virus Growth by Repeated Addition ofTrypsin. To verify that the abrogation of influenza virus accumulationin Vero cell cultures was due to the loss of trypsin activity in theculture medium, several experiments were performed in which trypsinconcentration was restored during influenza infection in Vero cells byrepeated additions of trypsin to the culture medium. This procedure ledto an increase of the virus production in the cultures infected with lowinput doses, thus ensuring high final yields despite the lowmultiplicity of infection, as shown in Table 2. The effect on viralaccumulation due to loss of trypsin activity was especially evident in50 cm³ flasks (Table 2) and 6-well plates with dense confluentmonolayers (Table 3), that is, in the conditions favoring a rapid lossof trypsin activity due to high numbers of Vero cells. In 6-well plateswith nonconfluent monolayers, and in 24-well plates, the loss of trypsinactivity and corresponding lower viral accumulation was much lessdramatic because here the multicycle growth of the virus was supportedunder standard conditions (Table 3). Thus, since Vero cells aresuggested to produce one or more factors that inactivate trypsinactivity, this inactivation was reduced (and viral growth increased) byproviding a relatively higher proportion of trypsin containing medium toVero cells in the culture dishes or flasks.

EXAMPLE 2 Evaluation of Influenza A Virus Replication in Vero CellCultures

Materials & Methods

Viruses. To identify a suitable high growth strain, Vero cells wereinfected with the A/England/1/53 (H1N1) (HG) strain of influenza virus,a reassortant containing the gene segments coding for the two surfaceglycoproteins (HA and NA) from A/England/1/53 (H1N1) and the remainingsix genes from A/PR/8/34 (H1N1). For the first four passages, the viruswas left to adsorb for 1 hour at 37° C., after which the monolayer waswashed twice with warm phosphate buffered saline (PBS) solution toremove the unabsorbed viruses. Serum-free MEM with 0.3% BSA was thenadded; the maintenance medium contained TPCK-treated trypsin at 1.0μg/ml. The input dose of virus was 10-10⁻³ PFU/cell. The material forfurther passage was collected 72 hours postinfection (p.i.), withtrypsin (final concentration, 1.0 μg/ml) added at 48 hours p.i. Cellswere infected with serial 10-fold dilutions of viruses added to thewashed cell monolayer without previous adsorption. Virus accumulationwas estimated by visual determination of the CPE and HA titration ofculture fluid at different times p.i. (24, 48 and 72 hours). Infectivitytitrations were performed in 96-well plates. Tissue culture infectiousdoses (TCID₅₀ /ml) and egg infectious doses (EID₅₀ /ml) values werecalculated by the formula of Karber (Arch. Exp. Path. Pharmak.162:480-483 (1931)).

Virus-containing culture fluids were concentrated in an Amicon systemand purified by differential sedimentation through 25-70% sucrosegradients. Whole virus protein estimates were made by the method ofBradford (1976). To determine the yield of HA protein in viruses grownin Vero and MDCK cells, the virus proteins were separated by gradient(4-10%) SDS-PAGE and intensity of Coomassie blue-stained protein bandswas quantitated by densitometry.

Virus isolation from clinical material. Influenza A viruses wereisolated from the throat washings of patients with clinical signs ofinfluenza and collected in PBS to which 0.7% BSA was added. Cellcultures (both Vero and MDCK) or embryonated chicken eggs were infecteddirectly with freshly collected (not frozen) throat washings. Chickeneggs were inoculated amniotically and allantoically. Clinical samplesused for isolation were inoculated undiluted and at 10⁻¹ and 10⁻²dilutions and incubated for 72-96 hours. Trypsin was added at 0 and 48hours p.i. (1.0 μg/ml) and virus yield tested for virus replication withchicken and guinea pig erythrocytes. Each sample was given at least twopassages in chicken eggs or cell culture before being considerednegative.

Immunological tests. Monoclonal antibodies to the A/Baylor/5700/82(H1N1) and A/Baylor/11515/82 (H1N1) strains were prepared by the methodof Kohler & Milstein, Eur. J Immunol. 6:511-519 (1976). Polyclonalantisera to influenza A/England/1/53 virus (20 passages in Vero cells)were prepared in chickens by intravenous injection of virus-containingculture fluid. HA and HI reactions were performed in microtiter plateswith 0.5% (v/v) chicken erythrocytes. Guinea pig erythrocytes 0.5% (v/v)were used to test for the presence of primary influenza A isolates fromthe 1993-1994 winter epidemic season.

Gene amplification. RNA was isolated by treating allantoic or culturevirus containing fluids with proteinase K and sodium dodecyl sulfate andthen extracting the product with chloroform:phenol (1:1) and ethanolprecipitation as previously described (Bean et al., (1980)). Viral RNAwas converted to cDNA with the use of U12 (5'AGCGAAAGCAGG3')(SEQ IDNO:1) and AMV reverse transcriptase. The sequences of theoligonucleotide primers used in this study for molecularcharacterization of the internal genes (PB2, PB1, PA, NS and M) arereadily available.

Amplification proceeded through a total of 35 cycles of denaturation at95° C. (1 min), annealing at 50° C. (1 min.), and primer extension at74° C. (3 min.). Amplified DNAs were analyzed by electrophoresis,visualized with ethidium bromide and then purified with either the MagicPCR Preps DNA purification system™ (Promega, Madison, Wis.) or theGeneclean R Kit™ (BIO 101, La Jolla, Calif.) according to themanufacturers' instructions.

Nucleotide sequence determination. Nucleotide sequencing was performedby the dideoxynucleotide chain termination method with the Fmol™ DNAsequencing system (Promega). The reaction products were separated on 6%polyacrylamide-7M urea gels, 0.4 mm thick.

Results

Screening of influenza A viruses in Vero cells. Influenza viruses canreplicate to high titers in a limited number of cultured mammaliancells, provided that trypsin is present for cleavage of the HA molecule.To determine whether Vero cells were a suitable alternative system tochicken eggs for replication of influenza A viruses, a virus repositorywas screened for selection of a master strain that would replicatesufficiently in the mammalian epithelial-like cell line. MDCK cells,which are widely used to isolate and culture viruses, were included inthe study as a reference.

The influenza A virus strains that were examined had been isolated froma wide range of human and avian hosts, and represented 12 of the 14 HA(not H5 and H7) and 9 NA subtypes. Viruses were passaged three times inVero and MDCK cells with trypsin, and the virus yield was estimated fromHA and infectivity titers. Of the 72 strains investigated, 65 (90.3%)replicated to the level that can be detected by HA titration in Verocells after the first passage and 37 (51.4%) after the second. Bycomparison, all strains could replicate in MDCK cells during the firstand second passages. Six human viruses were selected as the strains withthe highest growth potential (Table 4), among which A/England/1/53(H1N1) (HG) virus was chosen for further adaptation to Vero cells tooptimize growth of virus in Vero cells.

If the A/England/1/53 (H1N1) (HG) virus is to be used as a master strainfor generation of high growth reassortants, it is necessary to establishthe genotype of this virus. Therefore, we partially sequenced the genesencoding the internal proteins and compared their nucleotide sequencewith the prototype influenza strain, A/PR/8/34 (H1N1). As shown in Table5, the A/England/1/53 (HG) strain selected for adaptation to growth inVero cells is itself a reassortant between the original A/England/1/53strain and A/PR/8/34. Six genes of the reassortant encoded internalproteins of A/PR/8/34 and two surface glycoproteins of A/England/1/53.

Infectivity of A/England/1/53 (HG) after serial passaging. To enhancethe yield of virus in Vero cells, we performed 20 serial passages ofA/England/1/53 (H1N1) (HG) at limiting dilutions, comparing the resultswith those for the parental strain (Table 6). Although the infectivityof the parent was lower in Vero cells than in either MDCK or chickenembryos, the progeny showed increased activity in Vero cells by thetenth passage, exceeding that in both reference systems. By thetwentieth passage, the infectivity of the virus was superior in Verocells in the continuous presence of trypsin, but the HA titers remainedcomparable (64-128).

The infectivity titer (TCID₅₀) was 26 times higher (6.95 vs 8.37) thanthat of the parental strain. By contrast, adaptation of replication inVero cells resulted in a slight attenuation of the virus when grown inchicken embryos, as indicated by a reproducible decrease in EID₅₀ titerfrom 8.2 to 7.7 log₁₀. The plaques formed by the Vero-adaptedA/England/1/53 (HG) influenza strain were not as clear in Vero as inMDCK cells, and the efficiency of production was 10-fold lower.Plaque-forming capacity in Vero cells increased during serial passagesof the virus but not in direct relation to the TCID₅₀ titers. Thus,after 20 serial passages in Vero cells in the continuous presence oftrypsin, the yield of infectious virus was high by comparison with thatin MDCK cells and embryonated chicken eggs.

Viral protein yield of influenza A/England/1/53 (HG) in Vero and MDCKcells. Viral protein yield is an important feature of any system used toproduce influenza virus vaccines. To establish the amount ofvirus-specific proteins that can be obtained from Vero cells, wecompared the protein yields of A/England/1/53 (HG) (20-passage) virusafter replication in Vero and MDCK cells (Table 7). Determination of theHA protein yield was done using SDS-PAGE separated virus proteins andwas quantitated by densitometry. Tests of culture fluids indicated thatapproximately 6×10⁸ of infected cells could produce 4.38 mg of virusprotein in Vero and 4.13 mg in MDCK cells. It was also possible toobtain viral proteins from disrupted, virus-infected cells of eithertype; the protein yields were lower than in the supernatant but therewas no significant difference between the cell types in amount of virusprotein.

Antigenic stability of the Vero-adapted influenza A/England/1/53 (HG)virus. Because repeated passage of influenza viruses in mammalian cellscould lead to changes in antigenicity, it was thought that it wasimportant to assess the influenza virus adapted to Vero cell culture forthis property. In HI tests with both polyclonal (chicken, rabbit andgoat) antisera to cross reacting influenza A (H1N1) viruses and withanti-HA monoclonal antibodies, there were no appreciable differences inHA reactivity between the parental strain of A/England/1/53 (HG) and itsserially passaged variants (Table 8). This finding, which extends toantibodies specific for H1N1 strains other than A/England/1/53 (HG),indicates that serial passage of the virus in Vero cells did not modifyits HA antigenic properties.

Primary isolation of influenza A viruses. Currently, MDCK cells providethe most sensitive host cell system for the primary clinical isolationof influenza viruses directly from patients. Vero cells have beensuccessfully used to isolate parainfluenza and mumps viruses, but theywere judged unsuitable for the isolation of influenza viruses. Toreassess this issue, we tested nine clinical specimens collected duringthe 1993-1994 epidemic season in three culture systems (Vero and MDCKcells and embryonated chicken eggs). Six influenza A (H3N2) strains wereisolated in Vero cells, seven in MDCK cells and only two in embryonatedchicken eggs (Table 9). Two samples failed to yield virus in any hostsystem. During the first passage in Vero cells, CPE (observed 48-72hours after inoculation) was the only evidence of virus reproduction. HAactivity was detectable on the 2nd passage, and by the 3rd passage thepositive samples produced both CPEs and HA titers that ranged from 2-32.In all three culture systems, it was necessary to use guinea pigerythrocytes to determine HA titers. Chicken erythrocytes failed to beagglutinated according to known methods. To examine whether replicationof influenza A (H3N2) viruses in Vero cells could select antigenicvariants, we analyzed viruses that had been passaged three times in thissystem. The reactivity patterns of the HA with polyclonal antisera toreference A (H3N2) influenza strains and monoclonal anti-HA antibodiesdid not indicate differences between the strains isolated in Vero cells.These results indicate that Vero cells would provide a useful and nearlyas sensitive a culture system as MDCK cells for primary isolation ofinfluenza A (H3N2) viruses, provided that trypsin was presentcontinuously in the Vero cell cultures.

EXAMPLE 3 Production of Influenza B Virus in Vero Cell Cultures

Materials and Methods

Cells. The Vero cell line was obtained from the American Type CultureCollection (ATCC, Rockville, Md.) at the 136th passage. Cells werecultivated as a monolayer at 37° C. and 5% CO₂ in Eagle's minimalessential medium (MEM) containing 10% unheated fetal calf serum (FCS),MDCK cells (London, Mill Hill) obtained at a low-passage level, weregrown in MEM with 5% heat-inactivated FCS. Plaque assays were performedwith TPCK-treated trypsin (2.5μg/ml; Worthington Diagnostics Freehold,N.J.) as described earlier (Hayden, F. G. et al., Antimicrob. AgentsChemother. 17:865-870 (1980)). Plaques were counted on the third dayafter infection.

Virus replication. The replicative properties of egg-grown influenza Aand B viruses, obtained from the repository at St. Jude Children'sResearch Hospital, were determined in Vero cells. The cells wereinfected with serial 10-fold dilutions (10¹ →10³) of virus in serum-freeMEM with 0.3% BSA, which was added to the washed monolayer withoutprevious incubation; the maintenance medium contained 1.0 μg/ml ofTPCK-treated trypsin added at 0,24 and 48 hr. postinfection. Virus yieldwas determined by HA and PFU titration of culture medium afterincubation for 72 or 96 hr. at 33° C. TCID₅₀ /ml and EID₅₀ /ml valueswere calculated as described by Karber, G., Arch. Exp. Path. Phannak,162:480-483 (1931).

Primary isolation. Vero and MDCK cells were infected with the throatcultures of patients with clinical signs of influenza. Only samples thatwere positive for influenza virus by indirect immunofluorescence test orby previous isolation in eggs or MDCK cells were used. Infected cellswere incubated for 72-96 hr. postinfection at 33° C. (for influenza Bviruses) or 37° C. (for influenza A viruses); trypsin was added at 0,24and 48 hr. postinfection (1.0μg/ml).

Adaptation to growth in the Vero cells. Adaptation to growth in Verocells was performed for the A/England/1/53 (H1N1) (HG) and B/AnnArbor/1/86 strains by 20 serial passages at limiting dilutions aspreviously described (Govorkova, E. A. et al., J. Infect. Dis.172:250-253 (1995)) and as described infra.

Antigenic analysis. Monoclonal antibodies to the HA of egg-grownA/Baylor/11515/82 (H1N1), A/Baylor/5700/82 (H1N1), B/Ann Arbor/1/86,B/Memphis/6/86 and MDCK-grown B/Memphis/6/86 viruses were preparedaccording to Kohler & Milstein, (Eur. J Immunol. 6:511-519 (1976)).Monoclonal anti-HA and polyclonal antibodies were used for antigeniccharacterization of the influenza A and B viruses in ahemagglutinin-inhibition (HI) reaction with 0.5% (v/v) chickenerythrocytes.

Analysis of the abundance of SA α2,3 Gal and SA α2,6 Gal linkages onVero and MDCK cells. This study was performed with the digoxigeninglycan differentiation kit (Boehringer Mannheim Biochemica; Germany).Briefly, Vero or MDCK cells were washed twice in PBS containing 10 mMglycine, then once with buffer 1 (50 mM Tris-HCI; 0.15M NaCl; 1 mM MgCl₂; 1 mM MnCl₂ ; 1 mM CaCl₂ ; pH=7.5). Digoxigenin (DIG)-labeled lectins,Sambus nigra agglutinin (SNA) specific for SA α2,6 Gal, and Maackiaamurensis agglutinin (MAA) specific for SA α2,3 Gal, were dissolved inbuffer 1 and then incubated with the cells for 1 hr. at roomtemperature. After three washes, the cells were incubated withanti-DIG-fluoresceinated, Fab fragments (1:40 diluted in buffer 1) for 1hr. at room temperature. After three washes, the cells were analyzed forrelative fluorescence intensity on a FACScan fluorospectrometer (BectonDickinson).

Surface immunofluorescence. Vero or MDCK cells were infected withA/England/1/53 (H1N1) or B/Ann Arbor/1/86 at different m.o.i.'s (0.1,1.0 and 10.0 PFU/cell). Cell suspensions were fixed at 6 hr.postinfection with 4% paraformaldehyde in PBS at room temperature for 20min. For detection of the HA of influenza viruses, we used a panel ofmonoclonal antibodies to the HA's of A/Baylor/5700/82 (H1N1) andB/Memphis/6/86. The cells were incubated with 1:100 dilution ofmonoclonal antibodies in PBS with 0.2% gelatin, 0.05% Tween 20 and 10.0%normal goat antisera for 1 hr. at room temperature. After threeextensive washes with PBS-0.05% Tween 20, the cells were incubated withfluorescein-conjugated goat anti-mouse antibodies (Sigma; 1:100dilution) for 1 hr. at room temperature. After three washes withPBS-0.05% Tween 20, the cells were analyzed on a FACScanfluorospectrometer.

Protein gel electrophoresis and radioimmunoprecipitation. Vero or MDCKcells were infected with either A/England/1/53 (H1N1) or B/AnnArbor/1/86 at an m.o.i. ˜30 PFU/cell. After adsorption for 1 hr. at 37°C. in virus growth medium, the cells were washed and incubated for 5 hr.(influenza A strain) or 8 hr. (influenza B strain) in the labelingmedium (DMEM without methionine and cystine; ICN Biomedicals, Inc.,Costa Mesa, Calif.). Viral proteins were radioactively labeled with Tran(³⁵ S) methionine/cysteine (ICN chemicals; 100 μCi/ml) for 2 hr. at 37°C. Viral proteins--HA, nucleoprotein (NP), and matrix (M1 and M2) wereanalyzed by radioimmunoprecipitation with specific monoclonalantibodies. The cell lysate (100 μl) was treated with 1-2 μl of specificvirus antibody and incubated for 2 hr. at room temperature with constantinverting. These experiments relied on monoclonal antibodies to HA ofA/Baylor/5700/82 (H1N1) virus, to NP of A/WSN/33 (H1N1) and to HA ofB/Memphis/6/86. Monoclonal antibodies to the M2 protein (14 C2) weregenerously provided by Dr. R. A. Lamb (Department of Biochemistry,Molecular and Cell Biology, Northwestern University, Evanston, Ill.). Arabbit anti-mouse protein A suspension of beads was added to thesupernatant, which then was incubated further for 2 hr. at roomtemperature. The immunocomplexes were pelleted and washed three timeswith RIPA buffer (50 mM Tris-HCI, pH 7.6; 150 mM NaCl, 0.05 v/v TritonX-100, 1.0% sodium deoxycholate, 1.0% SDS, 1 mM EDTA), heated at 100° C.for 5 min in 2× Laemmli loading buffer and analyzed by 15% SDSpolyacrylamide gel electrophoresis (PAGE) according to the method ofLaemmli, U. K., Nature 227:680-685 (1970). Imaging and quantitation wereperformed with a Molecular Dynamics phosphorimager and Image QuantSoftware™, respectively.

Gene amplification. RNA extraction, cDNA preparation and the polymerasechain reactions (PCR) were performed as previously described (Gubareva,L. V. et al., Virology 199:89-97 (1994)).

Nucleotide sequence determination. Nucleotide sequencing was performedby the dideoxynucleotide chain termination method (Sanger, F. et al.,Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) with the FMOL™ DNAsequencing system (Promega) and end-labeled primers.

Electron microscopy. Vero and MDCK cell monolayers were infected withVero-adapted influenza strains (A/England/1/53 (H1N1) and B/AnnArbor/1/86) at an m.o.i. of 0.001 PFU/cell. Infected and control cellmonolayers were fixed at 48 hr. postinfection in cacodylate-buffered2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated ingraded series of alcohols and embedded in Spurr low-viscosity embeddingmedium (Ladd Research Industries, Burlington, Vt.). Ultrathin sectionsof cells were cut with a diamond knife on a Sorvall MT 6000ultramicrotome, and the sections were examined in a Phillips EM 301electron microscope operated at 80 kV.

Immunohistochemical assay. To detect apoptotic changes in virus-infectedVero and MDCK cells, we used a commercial kit (Apoptag™; Oncor,Gaithersburg, Md.) according to the manufacturer's instructions.

Thus, the Vero cell line appears to offer useful system for primaryisolation of influenza A and B viruses and could support virusmultiplication to high infectivity titers.

Results

Primary Isolation and replication of influenza A and B viruses in Verocells. High rates of primary isolation of influenza A virus fromclinical specimens (n=27) were obtained with Vero and MDCK cells but notwith eggs: 70.4%, 74.1% and 14.8%, respectively (Table 10). By contrast,the percentage of specimens (n=21) yielding influenza B virus wascomparable in Vero cells (47.6%) and eggs (42.9%) but higher in MDCKcells (57.1%). Ranges of TCID₅₀ titers were essentially the same whetherviruses were grown in Vero or MDCK cells or eggs (Table 10).

Antigenic and genetic stability of the HA of influenza strains isolatedand grown in different host cell systems. To determine whetherreplication of influenza viruses in Vero cell line selects hostcell-mediated HA variants, we performed antigenic and sequence analysesof the HA molecule. HI testing with polyclonal and anti-HA monoclonalantibodies revealed that the egg-grown B/Ann Arbor/1/86 strain wasantigenically stable in Vero cells, as it retained its originalantigenic characteristics during 20 passages in the mammalian host-cellsystem (Table 11).

It was also important to determine if growth in Vero cells selectedvariant virus populations during primary isolation. We thereforecompared the HA sequence of influenza B viruses (B/Memphis/1/93)isolated in Vero or MDCK cells or chicken eggs. The results indicatedamino acid substitutions in virus grown and isolated in chicken eggs, incontrast to findings in Vero- and MDCK-grown viruses (Table 12). Thischange involved residues 196 (Asp→Asn) and 198 (Ala→Thr) at the head ofthe HA molecule. The HA1 region of influenza B/Memphis/1/93 virusisolated and passaged in Vero cells was indistinguishable from that inthe MDCK-grown counterpart.

Thus, the similarity of antigenic and molecular characteristics ofinfluenza A and B viruses grown in Vero cells to MDCK grown virussuggests that this host system would generate virus populationsanalogous to those found in naturally infected hosts.

Receptor specificity of Vero cells and efficiency of influenza virus Aand B infection. Human influenza viruses bind preferentially to SA α2,6Gal linkages and viruses from lower mammals to SA α2,3 Gal (Baum &Paulson, Acta Histochem. Suppl. 40:35-38 (1990)). To characterize thenature and distribution of influenza virus-binding receptors on thesurface of Vero cells, we used indirect immunofluorescence microscopywith different lectins specific for either N-acetylneuraminic acid α2,3galactose (NeuAc α2,3 Gal) or N-acetylneuraminic acid α2,6 galactose(NeuAc α2,6 Gal).

The Maackia amurensis agglutinin (MAA), which is specific for NeuAc α2,3Gal, binds strongly to the surface of Vero cells, whereas the Sambucusniigra lectin (SNA), which recognizes NeuAc α2,6 Gal, binds only weaklyto the surface of Vero cells. Both types of lectins bind strongly toMDCK cells. In these experiments, FACS analysis revealed that thereceptor specificity on Vero cells was predominantly NeuAc α2,3 Gal(˜90.0%), with a minority of cells also possessing the NeuAc α2,6 Gallinkage (˜21.0%). This result contrasts with the nearly equalproportions of these linkages found on more than 85% of the MDCK cellsexamined (FIG. 1). Thus, the Vero cell population probably comprises twodistinct groups of receptor-bearing cells, one containing both types ofsialyloligosaccharides specific for influenza viruses and the otherprimarily NeuAc α2,3 Gal.

Conceivably, the limited availability of NeuAc α2,6 Gal linkages on Verocells could affect the efficiency of infection by influenza A and Bviruses. We tested this prediction by determining the percentage ofcells expressing the HA molecule at different m.o.i.'s at 6 hr.postinfection (Table 13). The results of FACS analysis showed similarefficiencies of infection between Vero and MDCK cells infected witheither influenza A or B viruses. At an m.o.i. of 10.0 PFU/cell, 87% ofVero and 91% of MDCK cells were infected with the influenzaA/England/1/53 strain. Slightly smaller percentages of cells expressingthe HA were infected by the influenza B/Ann Arbor/1/86 virus: 81% forVero and 85% for MDCK (Table 13). Similar correlations were observedbetween Vero and MDCK cells at m.o.i.'s of 0.1 and 1.0 PFU/cell.

These results indicate that although human influenza viruses have apreference for α2,6 Gal-linked sialic acid, they are still able toinfect and replicate efficiently in Vero cells, where the primarylinkage is NeuAc α2,3 Gal as long as trypsin is continuously present inthe culture medium.

Protein synthesis in Vero and MDCK cells infected with influenza A and Bviruses. We also thought it important to assess the pattern of proteinsynthesis of influenza viruses grown in Vero versus MDCK cells. Here weanalyzed the protein synthesis in Vero cells infected with eitherinfluenza A/England/1/53 or B/Ann Arbor/1/86 as compared to infection inMDCK cells. The protein patterns of A/England/1/53 infected Vero cellsdemonstrated that most virus specific polypeptides are synthesized inproportions similar to those in MDCK cells (FIG. 2A). There were nodifferences in electrophoretic migration of viral proteins synthesizedin both cell lines infected with parental or Vero-adapted influenzaA/England/1/53 strain (results not shown). Under the conditions of theseexperiments, M1 and NS1 migrated close to each other, so that additionalresolution of immunoprecipitates obtained with mouse anti-M1 monoclonalantibodies was attempted using SDS-PAGE (FIG. 2A).

Relative amounts of viral proteins synthesized in MDCK and Vero cellsinfected with influenza A virus are reported in Table 14. After 5 hr.postinfection, the two cell types contained similar proportions of NP,M2 and NS2, while approximately 10% more HA and 10% less M1/NSI weredetected in Vero compared to MDCK cells. To determine which protein (M1or NS1) was under-produced in Vero cells, we also analyzed the materialimmunoprecipitated from infected cells with anti-M1 monoclonalantibodies. The results (not shown) demonstrated a slightly lower amountof M1 protein in Vero cells (9.2% vs 14.7% in MDCK cells). As shown inFIG. 2B and also Table 14, the pattern of protein synthesis andproportions of the HA, NP, M1 and NS1 proteins were similar in Vero andMDCK cells infected with influenza B/Ann Arbor/1/86 strain.

Ultrastructural features of virus-infected Vero cells. To determine ifinfluenza virus-infected Vero cells show the same morphological changesas other polarized epithelial cells, we studied the ultrastructuralfeatures of these cells in comparison to MDCK cells, after infectionwith the A/England/1/53 and B/Ann Arbor/1/86 Vero-adapted influenzastrains. At an m.o.i. of 0.001 PFU/cell, both types of cells showednuclear and cytoplasmic inclusions typical of influenza virus-infectedcells, as well as numerous budding virions (FIGS. 3A-B). As in MDCKcells, influenza A and B virions were released from the apical surfaceof Vero cells, a feature typical of epithelial cells infected withinfluenza virus. The budding virions in both Vero and MDCK cellsappeared to be mainly filamentous. A portion of influenza A-andB-infected cells in both systems showed cytopathological changesindicative of apoptosis (FIGS. 3C-F, also Wyllie, A. H. et al., Inter.Rev. Cytol. 68:251-306 (1980)). The nuclear changes consisted ofblebbing of the nuclear envelope and condensation of the chromatin. Thecytoplasmic changes consisted of extensive vacuolation, blebbing andvesiculation of the plasma membrane to form "apoptotic bodies."

To confirm that our electron microscopic observations were indeedconsistent with cell apoptotic changes, we examined infected Vero andMDCK cells with an assay that detects fragmented DNA in the cells. Theresults were positive for 20% to 30% of the infected cells, contrastedwith none of the uninfected cells (not shown). This range of positivitymay be underestimated, as many positive cells could have detached fromthe substratum during the extensive washing required by theseprocedures. In certain cells, the fluorescent label was clearly seenover spherical masses within the nucleus, which may represent condensedmasses of degraded DNA. Thus, a substantial portion of infected Vero andMDCK cells undergo endonucleolytic cleavage of DNA--a feature typicallyseen in other types of cells infected with influenza virus (Takizawa, T.et al., J. Gen. Virol. 74:2347-2355 (1993); Hinshaw, V. S. et al., J.Virol. 68:3667-3673 (1994)).

Discussion. Several lines of evidence from the present study support theuse of Vero cells as a host system for cultivation of influenza A and Bviruses for vaccine production when trypsin is continuously present inthe culture system: (I) efficiency of primary virus isolation andreplication to high infectivity titers, (ii) genetic stability of the HAmolecule with maintenance of antigenic properties characteristic ofviruses derived from humans, (iii) similarities in the pattern ofprotein synthesis and morphological changes between virus-infected Veroand MDCK cells. Previous attempts to grow influenza virus in Vero cellswere met with limited success (Demidova, S. A. et al., Vopr. Virosol(Russian) 346-352 (1979); Nakamura & Homma, J Gen. Virol. 56:199-202(1981); Valette, M. et al., Antimicrob. Agents Chemother. 37:2239-2240(1993)). Moreover, when Vero cells were infected with A/fowlplague/Rostock/34 (FPV, H7N1), very little infectious virus wasreleased, and its spread was greatly impeded (Lau & Scholtissek,Virology 212:225-231 (1995)). It is now known that Vero cells rapidlydestroy exogenous trypsin (Kaverin & Webster, J. Virol. 69:2700-2703(1995)), limiting the replication of infectious viruses to a singlecycle. It is now discovered that the stepwise addition of trypsin cancircumvent the problem of trypsin inactivation or viral replicationinhibition and ensure multicycle replication comparable in all respectsto that seen with both human influenza A and B viruses grown in MDCKcells.

We also show that the HA1 region of influenza B viruses isolated andpassaged in Vero cells is indistinguishable from that of MDCK-growncounterparts, supporting the conclusion that Vero cells do not selecthost cell-mediated HA variants. Similar correlations were reported byKatz & Webster, J. Gen. Virol. 73:1159-1165 (1992) for the HAs ofinfluenza A (H3N2) viruses isolated in LLC-MK2 and primary guinea pigkidney cells and those of MDCK cell-grown viruses isolated from the samepatient. The absence of host cell-specific modifications of the HAduring primary isolation and subsequent passage in Vero cells is acritical requirement in validation of this cultivation system for theproduction of human influenza vaccines and diagnostic reagents. Aminoacid substitution at positions 196-198, near the tip of the HA molecule,is known to be involved in host cell-mediated mutations (Robertson, J.S. et al., Virology 143:166-174 (1985); Robertson, J. S. et al.,Virology 179:3540 (1990)). Further, the loss of a potentialglycosylation site by egg-grown viruses was associated with alterationsin binding of mono- and polyclonal antibodies to the HA molecule(Oxford, J. S. et al., J. Gen. Virol. 72:185-189 (1991)) and withattenuation of virulence for volunteers (Oxford, J. S. et al., Arch.Virol. 110:37 -46 (1990); Zuckerman, M. A. et al., J. Infect. Dis.28:41-48 (1994)). Thus, the antigenic and nucleotide sequencesimilarities between the HAs of Vero- and MDCK-grown influenza B virusesprovide reassurance against the generation of undesirable variants.

Receptor specificity is an important mechanism governing thesusceptibility of cells to virus infection. In the absence of sialicacid receptors of the proper specificity, viruses may be unable to bindto the cell surface, thus eliminating the opportunity for productiveinfection. Although Vero cells bore a relatively low level of the NeuAcα2,6 Gal linkage, by comparison to MDCK cells, this deficiency did notappear to affect their susceptibility to either influenza A or B virus(Table 13). This finding raises the possibility that linkages other thanNeuAc α2,3 Gal and NeuAc α2,6 Gal may be involved in the attachment ofinfluenza viruses to host cells.

Effective virus replication depends on specific cellular requirements,including the synthesis, transport and processing of viral proteinsneeded to produce infectious virus. Although, in the present study, theamounts of HA and M1/NS1 synthesized by Vero and MDCK cells differed byan estimated 10%, this discrepancy did not affect the virus yield. Thisobservation is consistent with data reported by Nakamura et al., J. Gen.Virol. 56:199-202 (1981) showing that the synthesis of M protein isselectively inhibited in Vero cells infected with influenza B/Lee/40virus. By contrast, overproduction of M2 and NS2 proteins in Vero cellsinfected with A/FPV (H7N1) was recently described by Lau & Scholtissek,Virology 212:225-231 (1995). In any event, the amount of M1 produced inVero cells seems to be sufficient to facilitate the nucleocytoplasmictransport of the nucleocapsid and the production of infectious virus.

Ultrastructural examination of influenza virus-infected Vero cellsrevealed morphological changes similar to those observed in MDCK cells.It is interesting that both cell lines produce predominantly filamentousviruses, which may explain the detection of lower HA levels than mightbe expected from the high infectivity rates associated with these hostcell systems. Although influenza virus can induce apoptosis in othercell lines (Takizawa, T. et al., J. Gen. Virol. 74:2347-2355 (1993);Hinshaw, V. S. et al., J. Virol. 68:3667-3673 (1994)) the observationsreported here provide the first morphological and cytochemical evidenceof this effect in Vero cells.

The MDCK cell line has been widely touted as the optimal mammalian cellsystem for the isolation and growth of influenza viruses, but it has notyet been approved for use in vaccine production. A report that MDCKcells can induce tumors in nude mice raises questions about thesuitability of this system for the production of live influenza virusvaccines. Thus, the Vero cell line grown in the continuous presence oftrypsin offers an attractive alternative for the cultivation ofinfluenza A and B viruses. This system is already in use for theproduction of other human virus vaccines and could readily be adapted toinfluenza viruses.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

                                      TABLE 1                                     __________________________________________________________________________    Accumulation of Influenza A/Rome/49 (HIN1) virus in MDCK cells and in         Vero cells with trypsin in different plasticware                                        Input dose (TCID.sub.50 per flask or per well)                      Plasticware                                                                             10,000 1,000  100    10                                             Cells                                                                             h.p.i..sup.1                                                                        48  72 48  72 48  72 48 72                                          __________________________________________________________________________    MDCK                                                                              50 cm.sup.3 flasks                                                                  32.sup.2                                                                          128                                                                              64  128                                                                              32  128                                                                              16 128                                         Vero                                                                              50 cm.sup.3 flasks                                                                  16   32                                                                               2   8  1   1  0  0                                          Vero                                                                              96-well                                                                             ND   16                                                                              ND   16                                                                              ND   32                                                                              ND  32                                             plates                                                                    __________________________________________________________________________     .sup.1 Hours postinfection (h.p.i.)                                           .sup.2 Reciprocals of HA titer in culture fluid                               ND = not done                                                            

                  TABLE 2                                                         ______________________________________                                        Effect of the restoration of trypsin concentration on X-31 virus              grown in Vero cells in 50 cm.sup.3 flasks                                     Inputdose(TCID.sub.50 /flask)                                                                1.5 × 10.sup.5                                                                    1.5 × 10.sup.3                                                                    1.5 × 10                             h.p.i..sup.1   48     72     48   72   48   72                                ______________________________________                                        Trypsin added at 0 h.p.i.                                                                     8.sup.2                                                                              8      1    1   0     0                                Trypsin added at 0, 12, 24,                                                                  64     128    32   128  4    128                               and 48 h.p.i.                                                                 ______________________________________                                         .sup.1 Hours postinfection                                                    .sup.2 Reciprocals of HA titers                                               TPCK  trypsin added to final concentration of 1.0 g/ml                   

                                      TABLE 3                                     __________________________________________________________________________    Effect of multiple trypsin addition on the growth of influenza A viruses      in Vero cells in plates                                                                                Input dose: (TCID.sub.50 per well)                                           10.sup.4                                                                           10.sup.3                                                                          10.sup.2                                                                           10.sup.1                                Exp.       State of the                                                                         Trypsin  7 4     7  4                                       No.                                                                              Virus                                                                             Plates                                                                            monolayer                                                                            added at:                                                                            48                                                                              2 8 72                                                                              48                                                                              2  8 72                                    __________________________________________________________________________    1  FW/50                                                                             6-well                                                                            Dense  0 h.p.i..sup.1                                                                       64                                                                              6 3 64                                                                              2 2  1  1                                                              2                                                                              4 2                                                   FW/50                                                                             6-well                                                                            Dense  0, 24, 48 h.p.i.                                                                     64                                                                              6 3 64                                                                              16                                                                              3  8 32                                                               4 2     2                                             X-31                                                                              6-well                                                                            Dense  0. h.p.i.                                                                            64                                                                              6 1 16                                                                               1                                                                              2  0  1                                                               4 6                                                   X-31                                                                              6-well                                                                            Dense  0, 24, 48 h.p.i.                                                                     32                                                                              6 3 64                                                                              16                                                                              6  4 32                                                               4 2                                                2  FW/50                                                                             6-well                                                                            Non-confluent                                                                        0. h.p.i.                                                                            16                                                                              1 1 32                                                                              16                                                                              6  4  8                                                               6 6     4                                             FW/50                                                                             6-well                                                                            Non-confluent                                                                        0, 24, 48 h.p.i.                                                                     16                                                                              1 1 32                                                                              16                                                                              6  4 32                                                               6 6     4                                          3  FW/50                                                                             24-well                                                                           Non-confluent                                                                        0. h.p.i.                                                                            32                                                                              3 3 32                                                                              16                                                                              6  4 32                                                               2 2     4                                             FW/50                                                                             24-well                                                                           Non-confluent                                                                        0, 24, 48 h.p.i.                                                                     32                                                                              3 3 32                                                                              16                                                                              3  8 32                                                               2 2     2                                             X-31                                                                              24-well                                                                           Non-confluent                                                                        0. h.p.i.                                                                            64                                                                              6 3 64                                                                              16                                                                              6  4 32                                                               4 2     4                                             X-31                                                                              24-well                                                                           Non-confluent                                                                        0, 24, 48 h.p.i.                                                                     64                                                                              6 3 64                                                                              16                                                                              6  8 64                                                               4 2     4                                          __________________________________________________________________________     .sup.1 Hours postinfection                                                    .sup.2 Reciprocals of HA titer in culture fluid                          

                  TABLE 4                                                         ______________________________________                                        Highest Yields of Influenza A Viruses in Vero Cells                                                    HA      Infectivity titer.sup.1                      Virus Subtype                                                                            Virus         Titer.sup.1                                                                           (log.sub.10 TCID.sub.50 /ml)                 ______________________________________                                        Human  H1N1    A/Bellamy/42  32    7.9                                        viruses        A/Rome/49     64    7.7                                                       A/FW/1/50     64    7.6                                                       A/England/1/53 (HG)                                                                         64    7.5                                               H2N2    A/Japan/305/57                                                                              32    7.7                                                       A/Netherlands/65/63                                                                         64    7.6                                        ______________________________________                                         .sup.1 Titers were determined for the first passage and measured at 72        hours postinoculation.                                                        HG = High Growth                                                         

                  TABLE 5                                                         ______________________________________                                        Genotyping of A/England/1/53 (H1N1) Influenza Virus                           Genes and nucleotides analyzed                                                      PB2     PB1                                                                   969-    360-                                                                  1156    535     PA     NP    M    NS                                          1480-   1352-   20-227 1064- 506- 30-  H   N                            Virus 1580    1487    478-606                                                                              1246  881  205  A   A                            ______________________________________                                        A/PR/8                                                                              P       P       P      P     P    P    P   P                            34                                                                            A/Eng/1                                                                             E       E       E      E     E    E    E   E                            /53                                                                           (ori-                                                                         ginal)                                                                        A/Eng/1                                                                             P       P       P      P     P    P    E   E                            /53                                                                           (HG)                                                                          ______________________________________                                         P = genes from A/PR/8/32; E = genes from original A/England/153; HG = Hig     Growth The origin of HA and NA was determined by HI and NI tests.        

                  TABLE 6                                                         ______________________________________                                        Growth Characteristics of Influenza A/England/1/53 (H1N1) (HG)                Virus After 10 and 20 Passages in Vero Cells                                             Infectivity Titer.sup.1 (log.sub.10)                                                        Plaque-Forming                                                  TCID.sub.50 /ml   Units.sup.1                                                       MDC          EID.sub.50 /                                                                         (log.sub.10 PFU/ml)                      Virus   HA Titer K       Vero ml     MDCK  Vero                               ______________________________________                                        A/Eng/1/53                                                                            64       7.70    6.95 8.20   8.70  7.18                               (HG) parent                                                                   (1P)                                                                          A/Eng/1/53                                                                            64       7.20    7.95 7.70   8.57  7.76                               (HG) (10P)                                                                    A/Eng/1/53                                                                            64-128   7.57    8.37 7.70   8.65  7.70                               (HG) (20P)                                                                    ______________________________________                                         .sup.1 Measured 72 hours postinoculation                                      HG = High Growth; P = passages in Vero cells                             

                  TABLE 7                                                         ______________________________________                                        Viral Protein Yield of Influenza A/England/1/53 (H1N1) (HG) in Vero           and MDCK Cells                                                                                    Protein Yield.sup.1 (mg)                                  Host System                                                                              Material Investigated                                                                        Whole Virus                                                                             HA.sup.2                                  ______________________________________                                        Vero       Culture fluid  4.38      0.98                                                 Cells          1.25      0.36                                      MDCK       Culture fluid  4.13      1.18                                                 Cells          1.40      0.42                                      ______________________________________                                         .sup.1 The concentrated virus was derived from 1 liter of culture fluid o     sonicated infected cells; 6.1 × 10.sup.8 Vero and 5.8 ×           10.sup.8 MDCK cells were initially infected.                                  .sup.2 Quantitation of HA protein was determined by densitometry after        polyacrylamide gel electrophoresis.                                      

                                      TABLE 8                                     __________________________________________________________________________    Antigenic Analysis of Influenza A/England/1/53 (HG) Virus Passaged in         Vero Cells                                                                    HI Titers.sup.1                                                               Polyclonal antisera to:       Anti-HA monoclonal antibodies to:                    A/Eng/53                                                                            A/WSN/33                                                                            A/FW/1/50                                                                           A/USSR/90/77                                                                         A/Baylor/11515/82                                                                       A/Baylor/5700/82                      Virus                                                                              chick serum                                                                         rabbit serum                                                                        goat serum                                                                          rabbit serum                                                                         AB-32                                                                              AB-33                                                                              2/6                                                                              24/1                                                                             31/1                            __________________________________________________________________________    A/Eng/53                                                                           160   100   400   1600   400  3200 400                                                                              1600                                                                             200                             (HG)                                                                          A/Eng/53                                                                           160   100   200   1600   400  1600 400                                                                               800                                                                             200                             (2P)                                                                          A/Eng/53                                                                           160   100   400   1600   200  3200 400                                                                               800                                                                             200                             (7P)                                                                          A/Eng/53                                                                           320   100   200   1600   200  3200 400                                                                              1600                                                                             200                             (10P)                                                                         A/Eng/53                                                                           320   100   400   1600   200  3200 400                                                                              1600                                                                             200                             (20P)                                                                         __________________________________________________________________________     HG = High Growth; P = passage in Vero cells                                   .sup.1 Reciprocal of the highest dilution of antibody inhibiting 4 HA         units of virus                                                           

                  TABLE 9                                                         ______________________________________                                        Primary Isolation of Influenza A (H3N2) Viruses in Vero Cells During          1993-1994 Epidemic Season                                                     HA Titer at 48 and 72 Hours                                                   Postinfection in Vero Cells.sup.1                                             1st          2nd      3rd                                                     passage      passage  passage  Virus Isolation                                Strain  48     72    48  72   48  72   Vero MDCK.sup.2                                                                          Eggs.sup.3                  ______________________________________                                        A/Mem/1/93                                                                            <      <     2   2    8   16   +    +     +                           A/Mem/1/94                                                                            <      <     <   4    2    4   +    +     +                           A/Mem/2/94                                                                            <      <     <   4    <    8   +    +     -                           A/Mem/7/94                                                                            <      <     8   16   8   32   +    +     -                           Clinical                                                                              <      <     <   <    <   <    -    -     -                           Sample #1                                                                     01/12/94                                                                      A/Mem/11/                                                                             <      <     <   2    2   16   +    +     -                           94                                                                            Clinical                                                                              <      <     <   <    <   <    -    -     -                           Sample #2                                                                     01/12/94                                                                      A/Mem/12/                                                                             <      <     <   <    <   <    -    +     -                           94                                                                            A/Mem/16/                                                                             <      <     8   16   8   32   +    +     -                           94                                                                            ______________________________________                                         < = HA titer less than 1:2, + = virus isolated and typed as influenza A       (H3N2), - = virus not isolated                                                .sup.1 HA titration was performed with 0.4% (v/v) guinea pig erythrocytes     .sup.2 Virus was detected after the first passage in MDCK cells               .sup.3 Virus was detected on the second amniotic passage                 

                  TABLE 10                                                        ______________________________________                                        Primary Isolation of Influenza A and B Viruses                                from Clinical Samples                                                         Clinical Samples Yielding:*                                                   Influenza A virus (n = 27)                                                                        Influenza B virus (n = 21)                                       No. of HA                                                                              Infectivity No. of HA                                                                            Infectivity                                Cell   positive range       positive                                                                             range                                      System (%)      (log.sub.10 TCID.sub.50 /ml)                                                              (%)    (log.sub.10 TCID.sub.50 /ml)               ______________________________________                                        Vero   19       2.2-2.7     10     2.0-2.2                                           (70.4)               (47.6)                                            MDCK   20       2.8-3.1     12     2.7-3.0                                           (74.1)               (57.1)                                            Eggs   4        2.6-3.0     9      2.0-2.2                                           (14.8)               (42.9)                                            ______________________________________                                         *HA positivity and TCID.sub.50 values were determined at 72-96 hr.            postinfection after the second passage in the cell system tested.        

                                      TABLE 11                                    __________________________________________________________________________    Antigenic Analysis of Influenza B/Ann Arbor/1/86 Strain Passaged in Vero      cells                                                                                HI Titer                                                                                     Anti-HA monoclonal antibodies to:                              Polyclonal antisera to:                                                                      B/Hong Kong/8/73                                                                          B/Ann Arbor/1/86                            Virus  B/Vic/70                                                                           B/HK/73                                                                            B/AA/86                                                                            82/1                                                                              419/2                                                                             430/1                                                                             1/1 1/2                                     __________________________________________________________________________    B/AA/86(1P)                                                                           80  86   160  160 320 160                                                                              800  1600                                    B/AA/86(20P)                                                                         160  80   160  160 320 320                                                                              800  1600                                    __________________________________________________________________________

                  TABLE 12                                                        ______________________________________                                        Sequence Analysis of the HA1 Region of the Hemagglutinin of                   Influenza B Viruses Isolated and Passaged in Different Host Systems                                  Amino Acid                                                     Culture System   Position                                             Virus     Isolation  Passage Number                                                                            196-198.sup.1                                ______________________________________                                        B/Ann Arbor/1/86                                                                        Eggs       Eggs (n).sup.2                                                                            Asp--Thr                                                          Vero (20)   Asp--Thr                                     B/Memphis/1/93                                                                          Eggs       Eggs (n)    Asn--Ala                                                          Vero (10)   Asn--Ala                                               MDCK       MDCK (1)    Asn--Thr                                                          MDCK (3)    Asn--Thr                                               Vero       Vero (1)    Asn--Thr                                                          Vero (4)    Asn--Thr                                     ______________________________________                                         .sup.1 All nucleotide changes were restricted to codons for residues          196-198 within the HA1 region of each virus.                                  .sup.2 n = numerous passages in eggs.                                    

                  TABLE 13                                                        ______________________________________                                        Efficiency of Influenza A and B Virus Infection                               of Vero and MDCK Cells                                                               % Cell Expressing HA at 6 hr. postinfection                            Cell     at different m.o.i.'s                                                System   A/England/1/53 (H1N1)                                                                        B/Ann Arbor/1/86                                      moi      .1     1.0      10   .1     1.0  10                                  ______________________________________                                        Vero     2.9    45.2     87.1 1.7    16.7 81.2                                MDCK     8.7    45.4     91.0 2.6    25.7 85.1                                ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Relative Amounts of Influenza A and B Viral Proteins                          Synthesized in Vero and MDCK Cells*                                           Frequency Distribution of Proteins in Host Cells (%)*                         A/England/1/53 (H1N1)                                                                           B/Ann Arbor/1/86                                            Protein  Vero     MDCK    Protein Vero MDCK                                   ______________________________________                                        HA       32.6     22.1    HA      19.5 22.6                                   NP       34.1     33.2    NP      38.9 43.2                                   M1/NS1   21.8     31.4    M1      16.4 13.8                                   M2       5.8      6.9     NS1     25.2 20.4                                   NS2      5.7      6.4     NS2     ND   ND                                     ______________________________________                                         *The percentage of each influenza virus protein was calculated according      to the intensity of the band on a 15% SDSPAGE gel; the intensity of all       bands was taken as 100%.                                                      ND = Not done.                                                           

References

Air et al., Structure, Function, and Genetics 6:341-356 (1989)

Avery's Drug Treatment: Principles and Practice of Clinical Pharmacologyand Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987)

Barrett et al, Virology: A Practical Approach, Oxford IRL Press, Oxford(1985) pp. 119-150

Baum & Paulson, Acta Histochem. Suppl. 40:35-38 (1990)

Berkow et al., eds., The Merck Manual, 15th edition, Merck and Co.,Rahway, N.J. (1987)

Demidova et al., Vopr. Virosol (Russian) 346-352 (1979)

Dochmer, Dev. Biol. Stand. 68:33 (1987)

Ebadi, Pharmacology, Little, Brown and Co., Boston, Mass. (1985)

Edwards, J. Infect. Dis. 169:68-76 (1994)

Ewami et al., Proc. Natl. Acad. Sci. USA 87:3802-3805 (1990)

Frank et al., J. Clin. Microb. 10:32-36(1979)

Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y. (1990)

Govorkova et al., J. Infect. Dis. 172:250-253 (1995)

Grachev et al., In Guidance for the Production of Vaccines and Sera,Burgamov, ed., Medicine, Moscow (1978), p. 176

Grachev, In Zh. Microbiol. Epidemiol. Immunobiol. 2:76 (1987)

Gubareva et al., Virol. 199:89-97 (1994)

Hayden, F. G. et al., Antimicrob. Agents Chemother. 17:865-870 (1980)

Hinshaw, V. S. et al., J. Virol. 68:3667-3673 (1994)

Itoh et al Virus 18:214-226 (1968)

Itoh et al., Japan. J. Mol. Sci. Biol. 23:227-235 (1970)

Johnson et al, J. Cancer 24:27 (1979)

Karber, Arch. Exp. Path. Pharmak. 162:480-483 (1931)

Katz & Webster, J. Gen. Virol. 73:1159-1165(1992)

Katz & Webster, J. Infect Dis. 160:191-198 (1989)

Katz et al., J. Infect. Dis. 160:191-198 (1989)

Katz et al., J. Virol. 64:1808-1811 (1990)

Katz et al., Virology 156:386-395 (1987)

Katz et al., Virology 165:446-456 (1988)

Katzung, ed. Basic and Clinical Pharmacology, Fifth Edition, Appletonand Lange, Norwalk, Conn. (1992)

Kaverin & Webster, J. Virol. 69:2700-2703 (1995)

Kendal et al., Infect. Immun. 29:966-971 (1980)

Kerr et al., Lancet 1:291-295 (1975)

Kilbourne, Bull. M2 World Health Org. 41:643-645 (1969)

Kohler & Milstein, Eur. J Immunol. 6:511-519 (1976)

Kodihalli et al., J. Virol. 69:4888-4897 (1995)

Krug et al., The Influenza Viruses, Krug, R. M., ed., Plenum Press, NewYork (1989), pp. 89-152

Lau & Scholtissek, Virology 212:225-231 (1995)

Levinson et al., In Abnormal Cells, New Products and Risk, Hopps et al.,Tissue Culture Association, Galihesbury (1985), p. 161

Mahy et al., J. Biol. Stand. 5:237-247 (1977)

Mizrahi, ed., Viral Vaccines, Wiley-Liss, New York (1990)

Montagnon et al., Dev. Biol Stand. 47:55 (1987)

Monto, et al., J. Clin. Microb. 13:233-235 (1981)

Murphy, Infect. Dis. Clin. Pract. 2:174-181 (1993)

Muster et al., Proc. Natl. Acad. Sci. USA 88:5177-5181 (1991)

Nakamura et a., J. Gen. Virol. 56:199-202 (1981)

Ogra et al., J. Infect. Dis. 135:499-506 (1977)

Osol, ed., Remington's Pharmaceutical Sciences, Mack Publishing Co,Easton, Pa. (1980), pp. 1324-1341

Oxford et al., Arch. Virol. 110:37-46 (1990)

Oxford et al., J. Gen. Virol. 72:185-189 (1991)

Robertson et al., Biologicals 20:213-220 (1992)

Robertson et al, Giornale di Igiene e Medicina Preventiva 29:4-58 (1988)

Robertson et al., Virology 143:166-174 (1985)

Robertson et al., Virology 179:35-40 (1990)

Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)

Schepetink & Kok, J. Virol Methods 42:241-250 (1993)

Smith et al., J. Clin. Microbiol. 24:265 (1986)

Subbarao et al., J. Virol. 67:7223-7228 (1993)

Takizawa et al., J. Gen. Virol. 74:2347-2355 (1993)

Valette et al., Antimicrobiol. Agent and Chemotherapy 37:2239-2240(1993)

Wharton et al., in The Influenza Viruses, R. M. Krug, ed., Plenum Press,New York (1989), pp. 153-174

Wood et al., Virol. 171:214-221 (1989)

Wyllie et al., Inter. Rev. Cytol. 68:251-306 (1980)

Zuckerman et al., J. Infect. Dis. 28:41-48 (1994)

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGCGAAAGCAGG12                                                                __________________________________________________________________________

What is claimed is:
 1. A method for replicating at least one high growthstrain of a passaged clinical isolate, or reassortant, derived from atleast one naturally occuring mammalian influenza virus strain,comprising(a) infecting cultured primary mammalian cells with said highgrowth strain of said passaged clinical isolate or said reassortant toobtain infected cells; and (b) culturing said infected cells in thepresence of trypsin at a continuous concentration range of 0.05-1.0μg/ml in the culture medium during the influenza virus growth cycle, toobtain replicated mammalian influenza virus, wherein(i) when saidpassaged clinical isolates are used to provide said high growth strain,the passaged clinical isolates have not been passaged in avian eggs; and(ii) said cultured mammalian cells lack adventitous agents to the extentthat said cells are suitable to be certified for mammalian virus vaccineproduction.
 2. The method of claim 1, wherein the multiplicity of virusinfection of said isolate or said reassortant is 5×10⁻⁶ to 5×10⁻³TCID₅₀, said TCID₅₀ being the tissue culture infective dose sufficientto infect about 50% of the cultured cells per cell.
 3. The method ofclaim 1, wherein the multiplicity of infection of said isolates orreassortants is between 1×10⁻⁶ and 1×10⁻⁵ TCID₅₀ per cell.
 4. The methodof claim 1, wherein the trypsin is added at intervals in the culturemedium to maintain said trypsin concentration range during the viralgrowth cycle.
 5. The method of claim 1, wherein the trypsin iscontinuously added to the culture medium to maintain said trypsinconcentration range during the viral growth cycle.
 6. The method ofclaim 5, wherein the concentration of trypsin is maintained between 0.5and 0.9 μg/ml during the viral growth cycle.
 7. The method of claim 1,wherein the mammalian influenza virus is a strain of mammalian influenzaA virus.
 8. The method of claim 1, wherein the mammalian influenza virusis a strain of human influenza B virus.
 9. The method of claim 1,wherein said cultured mammalian cells are cultured primary epithelialcells or fibroblast cells.
 10. The method of claim 9, wherein saidcultured primary epithelial cells are Vero cells.
 11. The method ofclaim 10, wherein said Vero cells have a passage number of 20-190. 12.The method of claim 1, wherein said mammalian influenza virus is a humaninfluenza virus.